Evidence for DNA translocation by the ISWI chromatin-remodeling enzyme - PubMed
Evidence for DNA translocation by the ISWI chromatin-remodeling enzyme
Iestyn Whitehouse et al. Mol Cell Biol. 2003 Mar.
Abstract
The ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin-remodeling activities. Here, we studied the interaction of the ISWI protein with nucleosomal substrates. We found that the ability of nucleic acids to bind and stimulate the ATPase activity of ISWI depends on length. We also found that ISWI is able to displace triplex-forming oligonucleotides efficiently when they are introduced at sites close to a nucleosome but successively less efficiently 30 to 60 bp from its edge. The ability of ISWI to direct triplex displacement was specifically impeded by the introduction of 5- or 10-bp gaps in the 3'-5' strand between the triplex and the nucleosome. In combination, these observations suggest that ISWI is a 3'-5'-strand-specific, ATP-dependent DNA translocase that may be capable of forcing DNA over the surface of nucleosomes.
Figures

Binding of ISWI to nucleosomal and free DNA. (A) Gel shift assay of ISWI binding to nucleosomes with no overhanging DNA and 41 bp of extra DNA. Binding reactions contained 5% glycerol, 60 mM NaCl, and 0.5 nM nucleosomes. ISWI was present at 0 pmol (lanes 1 and 7), 0.125 pmol (lanes 2 and 8), 0.25 pmol (lanes 3 and 9), 0.5 pmol (lanes 4 and 10), 1 pmol (lanes 5 and 11), and 2 pmol (lanes 6 and 12). (B) Gel shift assays showing the binding of ISWI to DNA fragments of increasing length. Binding reactions contained 2.5% Ficoll, 120 mM NaCl, and 0.5 nM DNA. ISWI was incubated with twofold increments of ISWI ranging from 0.125 to 2 pmol as described for panel A. (C) Plot illustrating the binding of ISWI to 31-, 41-, and 77-bp DNA fragments. The fraction bound was calculated from the disappearance of free DNA.

Maximal stimulation of ISWI ATPase activity requires DNA outside the nucleosome. (A) TLC plate showing a time course of ATP hydrolysis with a nucleosome core and a nucleosome core with 41 bp of linker DNA. Reactions (20 μl) contained 11 pmol of ISWI, 9.5 pmol of nucleosomes, 100 mM NaCl, and 5 μM ATP. (B) Time course of ATP hydrolysis by ISWI in the presence of 9.5 pmol of nucleosomes with increasing sizes of linker DNA. 0A0 is a 147-bp nucleosome core derived from the MMTV NucA-positioning sequence, 0A23 is the same sequence with 23 bp of linker DNA attached to one side, and 0A41 has 41 bp of DNA attached to one side. DNA is a 208-bp fragment encompassing the NucA-positioning sequence.

ISWI ATPase activity is stimulated by double- and single-stranded DNA in a length-dependent fashion. (A) Resolution of the products of typical ATPase reactions by TLC; (B and C) levels of ATP hydrolysis following stimulation with double- and single-stranded DNA, respectively. Reactions (10 μl) contained 40 pmol of duplex DNA, 10 μM ATP, and 200 mM NaCl. As lower levels of ATP hydrolysis were obtained in the presence of single-stranded DNA (ss DNA), high levels of ISWI (66 pmol) were included in these reactions compared to those in the reactions with double-stranded DNA (ds DNA) (12 pmol). The data points plotted represent the average ATP hydrolysis after 1 h at 30°C for three separate reactions. Further details are given in Materials and Methods.

ISWI-driven triplex displacement in the absence of nucleosome movement. (A) High-resolution site-directed nucleosome mapping was used to determine that an end-positioned nucleosome was not relocated by the action of the dISWI protein. Nucleosomes that were either centrally located on the DNA fragment 61A61 (lanes 1 and 2) or end positioned on the fragment 0A75 (lanes 3 and 4) were subjected to site-directed nucleosome mapping following incubation with ISWI in the presence or absence of ATP as indicated. The centrally located nucleosome was relocated to either end of the DNA fragment following remodeling, but the end-positioned nucleosome was not significantly repositioned. ISWI was also unable to alter the position of end-positioned nucleosomes when the triplex binding sequences were inserted in the adjacent DNA (data not shown). (B) A series of constructs were prepared that contained an end-positioned histone octamer and a 22-nucleotide, P32-labeled TFO positioned at 10-bp intervals, progressively more distant to the edge of the nucleosome. Each template was incubated with dISWI in the presence or absence of ATP. (C) Graph illustrating that the level of TFO displacement decreased as the triplex DNA was positioned further from the nucleosome.

Strand-specific DNA lesions prevent ISWI-driven triplex displacement. DNA templates were prepared that contained nicks, 5- or 10-bp single-stranded gaps, in either of the two DNA strands. 5′-3′, the top DNA strand; 3′-5′, the bottom DNA strand (with the nucleosome placed on the left). Each of the DNA lesions began 5 bp from the edge of the nucleosome; in addition, each template contained a 32P-labeled TFO positioned 20 bp from the edge of the nucleosome. Each template was incubated with dISWI in the presence or absence of ATP for 30 min at 30°C.
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